This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goal of this research is to explore similarities and differences in the translational regulation of a subunit of RNA polymerase and a ribosomal protein operon across diverse prokaryotes. The rpoS gene, encoding the alternative sigma factor sigma s, which influences bacterial virulence, is translationally regulated as a response to environmental changes in both Escherichia coli and Borrelia burgdorferi, the causative agent of Lyme disease. The roles of B. burgdorferi rpoS mRNA sequence, length and secondary structure, and the roles of trans-acting regulators, in particular a small non-coding RNA molecule, on translational initiation events will be characterized. In E. coli, the four ribosomal proteins of the alpha operon are translationally repressed by ribosomal protein S4, one of the proteins encoded in the operon. In Prochlorococcus marinus, Synechococcus sp. WH8102 and B. burgdorferi, rpsD, encoding S4, maps outside of the operon, suggesting a different mechanism of regulation. Both in vitro assays, using cell free extracts, and in vivo expression assays will be used to identify the cis-acting and trans-acting factors important in the translational regulation of the alpha operon in these diverse bacteria and to elucidate their mechanism of translational regulation.